Cell density-dependent vitamin D effects on calcium accumulation in rat osteogenic sarcoma cells (ROS 17/2)

Rat osteogenic sarcoma cells have been used widely as a model system to study actions of 1,25(OH)2D3 and other hormones in osteoblastlike cells. However, some of the pleiotypic manifestations of hormones in these cells vary greatly dependent upon the cell population density and other conditions of culture. Therefore, we have studied the effect of cell density on the relationship between 1,25(OH)2D3 and the initial 45Ca accumulation in ROS 17/2 cells in order to establish conditions suitable for studying the effect of 1,25(OH)2D3 on calcium fluxes in these cells. Cells were grown in the presence and absence of 1,25(OH)2D3 for 48 hours and then incubated for 4 min in the culture medium containing 0.5 microCi/ml of 45CaCl2. In high population density cultures, 0.25-1.0 pg/ml of 1,25(OH)2D3 stimulated the intracellular accumulation of 45Ca (cpm/mg protein), whereas 80 pg/ml or higher concentrations inhibited accumulation of 45Ca. In low density cultures, concentrations less than 80 pg/ml had no effect, 80-120 pg/ml increased the intracellular accumulation, and as much as 200 pg/ml failed to show the inhibitory effect. These results indicate that the ROS 17/2 cell responses to 1,25(OH)2D3 are biphasic--low concentrations stimulating and high concentrations inhibiting 45Ca accumulation. The sensitivity of the cells to 1,25(OH)2D3 increases as the cell population density increases. These observations suggest that the culture density and dose-response relationship must be carefully defined in in vitro studies utilizing osteogenic cell culture systems.